This invention relates to improvements in the expression and secretion of heterologous proteins from eucaryotic cells. Complete citations for references cited herein are set forth immediately preceding the claims.
The information which determines the destiny of a secreted protein is contained in its primary structure, and much of this information may involve dictating appropriate post-translational modification and correct conformation. The steps in the exocytotic pathway of the processing and transit of membranespanning and secretory proteins in mammalian cells have been described (for reviews see Farquhar Ann Rev Cell Biol 1985; Kornfeld & Kornfeld Ann Rev Biochem 1985). A large body of work has shown that proteins destined for the cell surface are first cotranslationally translocated into the lumen of the endoplasmic reticulum (ER) mediated by a signal sequence at or near the amino terminus of the nascent chain (Blobel & Dobberstein J. Cell Biol 1975; Walter et al Cell 1984). Inside the endoplasmic reticulum the signal sequence is usually removed and a high mannose oligosaccharide core unit is transferred to a sparagine residues located in the sequence Asn-X-Ser/Thr where X can be any amino acid, except perhaps proline. This N-linked core glycosylation occurs cotranslationally and it appears that the efficiency of glycosylation is dependent on the presentation of an appropriate conformation of the peptide chain as it enters the endoplasmic reticulum. Potential N-linked glycosylation sites may no longer be accessible after the protein has folded (Kornfeld & Kornfeld).
Proteins move from the endoplasmic reticulum to the Golgi apparatus where modifications such as sulfation and processing of the high mannose oligosaccharide chain to a complex type occurs and the proteins are directed to their proper destinations (Dunphy & Rothman Cell 1985). The movement from the ER to the Golgi has been identified as the rate limiting step in intracellular transport (Lodish et al. Nature 1983; Fitting & Kabat JBC 1982 J. Cell Biol 1985). Few proteins resident in the ER have been extensively studied for their interaction with secretory proteins transiting that compartment.
Environmental stresses such as heat shock induce the synthesis in prokaryotic and eukaryotic cells of a set of highly conserved heat shock proteins. (Schlesinger, J. Cell Biol 1986). hsp70 is the most abundant of these induced proteins. Proteins related to hsp70 are found in unstressed mammalian cells. There are three main members of the mammalian hsp70-like group of proteins: hsp70, hsc70, and GRP78 (Pelham Cell 1986) Following heat shock, synthesis of hsp70 is induced and the protein migrates to the nucleus where it is found in tight association with nucleoli. hsp70 can be released from this association by the addition of ATP in vitro. It has been hypothesized that hsp70 disaggregates heat damaged proteins by an ATP dependent mechanism to facilitate recovery from heat shock (Lewis & Pelham EMBO J 1985). hsc70 is found at high basal levels in growing cells and is only slightly heat inducible (Pelham Cell 1986). hsc70 has recently been identified as "uncoating ATPase", a constitively expressed enzyme that releases clathrin triskelions from coated vesicles in an ATP dependent reaction (Chappell et al Cell 1986, Ungewickell EMBO J 1985).
GRP78 was initially reported to be one of two proteins whose synthesis was induced by glucose starvation in chick fibroblasts (Shiu et al Proc. Natl. Acad. Sci. U.S.A. Its synthesis can also be induced by inhibitors of N-linked glycosylation such as tunicamycin, glucosamine or 2-deoxyglucose (Olden et al Proc. Natl. Acad. Sci. U.S.A 1979, Pouyssegur et al Cell 1977). GRP78 is not heat inducible and its basal level is high in secreting cells. Recently it has been shown that GRP78 is similar if not identical to immunoglobulin heavy chain binding protein (BiP) (Munro and Pelham Cell 1986). GRP78 is therefore also referred to hereinafter as BiP/GRP78 or simply, BiP. BiP was first described for its association with immunoglobulin heavy chains in pre-B cells (Haas and Wable Nature 1983). BiP transiently complexes with immunoglobulin heavy chain in the endoplasmic reticulum of secreting hybridomas. When assembly with light chains occurs BiP dissociates from the complex. In the absence of light chains BiP remains associated with heavy chains and this complex is not transported from the endoplasmic reticulum to the Golgi apparatus (Bole et al J. Cell Biol 1986). These subcellular fractionation studies showed that BiP is predominantly localized to the endoplasmic reticulum. The heavy chain-Bip complex can be dissociated in the presence of ATP suggesting a functional analogy with the hsp70 complex in heat shocked nucleoli. (Munro & Pelham Cell 1986).
We believe that BiP/GRP78 may associate in secreting cells with underglycosylated or improperly folded proteins in the endoplasmic reticulum and help clear them in analogy to the hypothesized role of hsp70 in the nucleus (Pelham Cell 1986). Such a function is consistent with the induction of increased levels of GRP78 synthesis under conditions which disrupt N-linked glycosylation. Recent studies on abberant proteins which fail to transit out of the ER have been interpreted to show that BiP binds to them in the ER although the identity of grp78 and BiP was disputed (Gething et al Cell 1986; Sharma et al EMBO J 1985) and the extent and degree of such binding was not specifically characterized. BiP/GRP78 may also associate with partially assembled proteins and retain them in the ER until assembly and processing is complete as is the case for the processing of immunoglobulin heavy chain (Bole et al J Cell Biol 1986).
Independent of the research on BiP mentioned above, we have conducted extensive research on the production of glycoproteins, including Factor VIII, in genetically engineered host cells. In the course of this research we have surprisingly found that a significant proportion of Factor VIII produced in vitro, e.g. in CHO cells, is not secreted into the cell culture medium. We have now surprisingly found that secretion levels for Factor VIII and other glycoproteins can be decreased by providing higher intracellular levels of BiP and can be increased by reducing the intracellular BiP level.